Endotoxins, which are commonly known as pyrogens, are substances produced by microorganisms growing in water or in aqueous solutions. They cause inflammation and general fever when injected intravenously. They are colloidal in nature and they persist even after the organisms which produce them are destroyed by sterilization.
The pyrogens are classified into six groups:
______________________________________ GROUPS: GRAM NEGATIVE SPECIES ______________________________________ (1) Shigella group: S. flexneri (dysentery) (2) Serratia marcescens group (2 strains) (3) Salmonella group: S. abortus equi S. enteritidis S. typhosa 0901 S. typhimurium (4) Escherichia coli group: E. coli 0127:B8 E. coli 055:B5 E. coli 026:B6 E. coli 0111:B4 E. coli 0128:B12 (5) Pseudomonas group (6) Porteus group ______________________________________
Neither the mode(s) of their activity nor their structures are known. However, in general, it is known that in gram-negative bacteria, the glycopeptide basal layer is covered with lipopolysaccharide which constitutes 20-30% of the cell wall. Lipopolysaccharides, components of the whole somatic antigen, with molecular weights of the order of 10.sup.6 to 10.sup.7, exhibit endotoxin activity. It is difficult to construct a molecular model for a lipopolysaccharide which would adequately explain the shapes revealed by electron microscopy.
Endotoxins of high antigentic activity have been extracted from bacteria by using one of the recognized published procedures. The best known are the Boivin trichloroacetic acid procedure (Boivin, A., and Mesrobeanie, L., Compt. Rend. Soc. Biol. 113,490, (1933); 128,5 (1938); as modified by Webster, Sagin Landy and Johnson (Webster, M. E., Sagin, J. F., Landy, M. and Johnson, A., J. Immunology, 74, 455 (1955), and the trypsin digestion method of Hartwell and Shear (Hartwell, J. L., and Shear, M. J. J. Nat. Cancer Inst., 4, 107-22, (1943). These methods produce bacterial endotoxins possessing properties adequate for most immunological and pathological studies. The purified endotoxins are comparatively stable in the solid form, but they may become inactivated in solution by hydrolysis.
Shigella dysenteriae has been proposed as an international reference bacterium for preparing endotoxins for pyrogenic activity. The endotoxin derived from this source contains 4.5-4.6% nitrogen and 0.80-0.85% phosphorus. It has a molecular weight of about 10.sup.7 and a strong pyrogenic activity at a level of 0.01 ppm in water (Humphrey, J. H., and Bangham, D. R., Bull Org. Mond. Sante, 20 1241-44 (1959).
The first significant pyrogenic work was done between 1911-1916 by Jona (Jona, J. L., Med. J. of Australia iii, 71-73 (1916) and Hort and Penfield (Hort, E. C., and Penfield, W. J. Britt. Med. J., 2, 1589 (1911). They discovered that introvenous infusions cause elevation of body temperature. In their work freshly prepared distilled water was injected into both a man and animals as a control. A portion of the same distilled water was innoculated into an unsterile container for a period of time and this preparation was then injected into the same man and animals. The febrile reaction occurred only when the innoculated distilled water was injected into the unsterile container. The concluded that the fever-causing product, called pyrogen, was associated with bacteria but was not a part of the bacteria themselves since autoclaving, boiling and filtering did not reverse the febrile reaction. They also were able to classify microorganisms into pyrogenic-type gram negative and nonpyrogenic-type gram positive. Because of the lack of advanced equipment and knowledge their experiments were limited, but they formed the basis for the development of the modern standardized pyrogen test. Siebert (Siebert, F. B., Amer, J. Physiol. 67, 90-04 (1923) and 71, 621-51 (1925), in 1923, confirmed these observations by injecting distilled water into rabbits. In her experiments she demonstrated that bacterial products are present in all pyrogenic fluids that are unaffected by autoclaving, boiling and filtering techniques. She concluded that pyrogenic reactions occur even in sterile fluids and that pyrogenic reactions can be prevented in pharmaceutical preparations only by the removal of pyrogens.
In the conventional method parenteral solutions are examined for pyrogens by using rabbits as test animals according to the procedure outlined in the United States Pharmacopeia (USP). This procedure was adopted in the early 1940's when the need for an official pyrogen test was first recognized. A collaborative study was undertaken by the Food and Drug Administration, the National Institutes of Health, and 14 pharmaceutical manufacturing companies. As a result of these studies, the first official pyrogen test was adopted and appeared in the XIIth Revision of the United States Pharmacopeia. Pharmaceutical companies and research laboratories have made extensive use of the USP rabbit pyrogen test during the past 35 years because of its low cost and the ease with which the rabbits are handled during the test. However other animals such as dogs, cats, monkeys, and horses are equally reliable for the test. The rabbit is reported to be the most sensitive animal for indicating the absence of pyrogens, whereas the dog is the most sensitive animal for establishing the presence of pyrogens. Some investigators have used both animals in order to obtain a better indication, particularly in doubtful cases.
The test is performed in a room in which the temperature and humidity are maintained at the same levels as the room in which the animals are housed.
The rabbit test described in detail in application Ser. No. 745,966 now U.S. Pat. No. 4,093,381 and incorporated herein by reference.